Interpretive Summary: Although E. coli O157 is the best known E. coli pathogen in humans, recently non-O157 E. coli have been identified as significant additional risks for disease. Testing for O157 and the Shiga toxins produced by all E. coli pathogens is important for controlling the spread of these bacteria, and for tracing outbreaks. While individual tests for O157 and Shiga toxins exist, the cost and speed of testing limit their use by food producers. This paper describes a new walk-away testing system based on the Dynex DS-2™ Automated ELISA Processing System workstation. This system can simultaneously detect up to 10 different types of bacteria and toxins in a single sample, although in this paper we demonstrate a "proof of principle" by simultaneous testing for 3 factors: O157 and two types of Shiga toxin (Stx 1 and Stx2). This system reduces the amount of human labor required, which leads to increased speed and accuracy. Cost and sample size are reduced by performing all tests simultaneously on the same sample. Because each test is performed on a small plastic bead, the end-user can easily modify the system to test for toxins and bacteria of interest, by simply using different beads. Furthermore, this system can be used for detection of other materials, including specific proteins and antibiotics.

Technical Abstract:
Conventional enzyme linked immunosorbent assay (ELISA) is a gold standard for screening antibodies and testing for protein or antigen presence. A significant limitation of this assay resides in the fact that only one analyte can be assessed per microplate well. Here, we describe and investigate a new technology consisting of an automated ELISA system in which up to 10 analytes can be measured within one single well, thus improving productivity, accuracy and repeatability by reducing the amount of human labor required. Another strength of the platform is that a user can load any necessary sets/subsets of beads to perform required assays, with improved flexibility compared to manufactured-loaded arrays for multiplex analysis. We also demonstrate that this system can be used to determine the pathogenicity (i.e., presence of Shiga toxins) and serotype (i.e., E. coli O157) of E. coli isolates.